The present invention pertains to homogeneous immunoassay systems involving complement-mediated lysis of liposomes containing markers.
Liposomes are micron-sized spherical shells of amphipatic molecules which isolate an interior aqueous space from the bulk exterior aqueous environment. They can be made to contain hydrophobic molecules within their membrane, or hydrophilic markers within their internal aqueous space, or both. Because of this versatility, liposomes are of interest both as potential vehicles for the delivery of drugs in vivo and as the basis for immunoassay systems in vitro.
Various formats for liposome immunoassay systems have been developed including heterogeneous and homogeneous systems. Heterogeneous systems, which typically require an initial separation of bound and unbound forms of tracer, are described in O'Connell, et al., Clin. Chem., 31:1424-1426 (1985) and MacCrindle, et al., Clin. Chem., 31:1487-1490 (1985). Homogeneous methods, such as those based on i) complement-mediated lysis, ii) melittin-mediated lysis, iii) color changes induced by cation-responsive dyes in perturbed membranes, and iv) enhanced agglutination, have also been described. Some of these methods rely on liposomes to generally amplify immunological reactions whereas others rely on the utility of liposomes to encapsulate marker substances within the liposome and to subsequently release them in proportion to the amount of analyte present in a sample.
An immunoassay system of particular interest to the background of the invention is the Liposome Immuno-Lytic Assay (LILA) which involves the antibody-triggered complement-mediated lysis of liposomes. In an exemplary assay format, a liposome encapsulating a marker is first made immunoreactive by association of a first immunological binding pair member (e.g., an antigen) with its surface. The liposome is then incubated with a fluid sample to be analyzed for the presence of the corresponding binding pair member (e.g., an antibody). Typically, the binding of antibody to antigen (pre-bound to the liposome surface) generates a liposome immune complex and, upon the addition of serum, complement activation is initiated leading to lysis of the liposome and release of the internal marker substance. The amount of analyte present in the sample is proportional to the amount of marker substance released.
Liposome lysis can be detected in a variety of ways and depends upon the nature of the marker initially encapsulated within the liposome. Kataoka, et al., Eur. J. Biochem., 24:123 (1971), for example, describe Lipid A sensitized liposomes which release a spectrophotometrically detectable glucose marker when incubated with an anti-Lipid A anti-serum and complement source. Yet another means for detecting lysis involves initially encapsulating within the liposome a fluorophore at self-quenching concentrations. Upon liposome lysis, an extreme dilution of the fluorophore occurs and this dilution re-establishes fluorescence. The increase in fluorescence is proportional to the amount of analyte present in the sample. Ishimori, et al., J. Immuno. Methods, 75:351-360 (1984) describe an immunoassay technique using immunolysis of liposomes to measure antibody against protein antigens such as human IgG. The marker used was carboxyfluorescein and the technique was reported to be effective at detecting 10.sup.-15 mole of anti-human IgG antibody or human IgG. Similarly, Yasuda et al., J. Immun. Methods, 44:153-158 (1981), describe the utilization of complement-mediated immune lysis of liposomes entrapping carboxyfluorescein at self-quenching concentrations to measure anti-glycolipid antibody.
The use of antibody sensitized liposomes in Liposome Immuno-Lytic Assays presents a number of system design problems not present in assays employing antigen coupled liposomes. Of interest to the background of the present invention are references describing developments in the art relating to procedures for coupling antibodies to liposome surfaces (Heath, T.D. and Martin, F.J., Chemistry & Physics of Lipids, 40:347-358 (1986); Martin, F.J. and Kung, V.T., Annals New York Academy of Sciences, 446:443-456 (1985)) which describe binding characteristics of antibody-bearing liposomes; and especially those references which relate to avoidance of liposome aggregation--a phenomenon which can seriously limit the sensitivity of LILA's. As one example, Jou, et al., Fed. Proc., Fed. Am. Soc. E. Biol., 43:1971 (1984) disclose coupling procedures designed for avoidance of aggregation of antibody-sensitized liposomes through a series of steps including: (1) limiting the average number of reactive functional groups per antibody molecule to less than one; (2) providing for early "quenching" during the coupling reaction; and, (3) employing dialysis to remove uncoupled antibodies. In this reference however, only liposome aggregation as a result of antibody-liposome coupling was addressed and no information was provided regarding the use of antibody-coupled liposomes for immunoassays.
Umeda, et al., J. Immun. Meth., 95:15-21 (1986) (and Umeda, et al., Jap. Patent Appln. No. Sho 59 [1984-261806]) describe a series of studies regarding a complement-mediated liposome immune lysis assay using carboxyfluorescein-entrapped liposomes sensitized with antibody to C-reactive protein (CRP) antigen. Whole antibodies, derived from different animal sources, were modified and coupled to liposomes utilizing a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2 pyridyldithio)-propionate (SPDP) and dithiothreitol (DTT), a reducing agent. However, upon coupling certain antibodies, e.g., rabbit antibodies, to dithiopyridyl-substituted dipalmitoylphosphatidylethanolamine (DTP-DPPE) liposomes, complement activation and liposome lysis occurred even in the absence of sample containing analyte. The low level of complement reagent required to minimize this non-specific lysis necessarily lowered the overall sensitivity of the assay. In addition, only certain animal sources of complement, i.e., guinea pig serum, proved to be effective reagents and this effectiveness also depended upon the animal source of the antibody coupled to the liposome. For example, goat antibody was suitable as the antibody bound to the liposome, but was not suitable as the secondary antibody. Although the assay sensitivity was in proportion to the amount of antibody bound to the liposome, when more than 400 .mu.g of IgG/.mu.mol of lipid was bound to the liposomes, liposomes became fragile and their spontaneous release of carboxyfluorescein increased irrespective of the liposome lipid composition. The sensitivity of the assay was improved by purification of whole antibody (by passage through an affinity chromatography column) prior to binding to liposomes. However, sensitivity was not increased when Fab' antibody fragments (which were expected to be coupled to liposomes more efficiently than IgG) were bound to liposomes. The Fab' antibody fragments of the reference were prepared by reducing F(ab').sub.2 antibody fragments with mercaptoethylamine and were then coupled via thiol residues to derivatized liposomes containing DTP-DPPE. In the attempt to explain the lack of improved sensitivity over that obtained using liposomes bearing whole goat antibody, it was speculated that the affinity of Fab' antibody fragments for antigen may be reduced during the drastic pepsin digestion at pH 4.5. It was thus suggested that the use of "high affinity" Fab' fragments would result in a much higher sensitivity than use of whole IgG.
The coupling of Fab' antibody fragments to liposomes via a disulfide exchange reaction requires either a sulfhydryl reactive derivative on the liposome or a derivatization of the sulfhydryl group on the Fab'antibody fragment. For example, Martin, et al., Biochemistry, 20:4229 (1981), describes the use of N-[3-2-pyridyldithio)propionyl]phosphatidylethanolamine (PDP-PE) liposomes, in a disulfide exchange reaction. The sulfhydryl reactive derivative is a pyridyldithio derivative. Martin, et al., J. Biol. Chem. 257:286 (1982), describes the use of N-[4-(p-maleimidophenyl)-butryl]phosphatidylethanolamine (MPB-PE) liposomes having reactive maleimide moieties for forming an essentially irreversible antibody-vesicle linkage which did not involve the usual disulfide linkage but rather involved the more stable thioether linkage. The liposomes of the two Martin references did not contain an encapsulated fluorophore as these liposomes were intended for "targetting" use rather than for use in an immunoassay. Bredehorst, et al., Biochemistry, 25:5693-5698 (1986) describes the coupling of Fab' fragments to MPB-PE liposomes. These liposomes did contain encapsulated fluorophore but the liposomes were noted to release up to 95% of the entrapped fluorophore. To overcome this leakage problem, a decrease in the molar concentration of the MPB-PE anchor in the liposomes was required which caused a corresponding decrease in the number of Fab' molecules bound per liposome. No evidence was given as to whether such coupled liposomes would be functional in an immunoassay.
A number of references have described derivatization of Fab' antibody fragments for use in the preparation of bispecific antibodies--hybrid immunoglobulins provided with two different antigen-binding sites through a chemical re-association of monovalent fragments derived. See, e.g., Brennan, et al., Science, 229:81 (1985) and Paulus, H.P., PCT Patent Application WO 85/04811. Both references show the preparation of Fab' thionitrobenzoate derivatives in which arsenite is used as a complexing agent to stabilize vicinal dithiols and to impede intramolecular disulfide formation.
In sum, several immunoassay systems involving complement mediated lysis of marker-encapsulating, antibody bound liposomes have been described. However, none of these are homogeneous systems totally responsive to the need in the art for highly sensitive assays for antigens in fluid samples.